This invention relates to a method and assembly for assaying an anticoagulated whole blood sample for the presence or absence of circulating fragments of cancer cells or fragments of other target cells. The blood sample is contained in a transparent sampling container assembly and the assay can be performed in situ in the sampling tube assembly. More particularly, the method of this invention involves the centrifugal density-based separation of the contents of the blood sample in a manner which will ensure that any circulating target cell fragments in the blood sample are physically displaced by their density into a predetermined axial location in the blood sample and in the sampling container assembly, and also into a restricted optical plane in the sampling container assembly which is adjacent to the wall of the sampling container, and finally into a very well-defined zone of that optical plane.
Cytology is the science and technology involved in the morphological characterization of mammalian cells. Cytology has clinical utility in both human and veterinary medicine. Cytology is most often used to diagnose the presence or absence of malignancy in exfoliated or harvested cells: a) that are shed into a body cavity such as the pleural space or peritoneum; b) that are shed into a body fluid that is excreted as, for example, sputum or urine; c) that are obtained by scraping or brushing a body surface, such as the uterine cervix, the uterine cavity, or bronchial mucosa; or d) that are obtained by direct needle-mediated aspiration from a tumor such as tumors of the thyroid, breast, lung, or the like. The exfoliated or harvested cells are then typically fixed, stained and visually studied, usually by bright field microscopy, and then, if needed, by immunologic stains and/or other molecular techniques.
This year approximately five hundred sixty thousand people will die from solid tumors (predominantly carcinomas) in the USA. Many of these deaths could be prevented by early diagnosis of these malignancies. Unfortunately, with the possible exception of the Prostate Specific Antigen (PSA) test for prostate cancer, there is no practical and routine methods that have been found to be effective for early detection of solid tumors through blood analysis.
Through early detection of cervical cancer, the Pap smear has decreased mortality from cervical cancer in the United States by over seventy percent. Development of an analogous test for other solid tumors could have a similar impact on overall cancer mortality.
The presence of circulating cancer cells that are spontaneously shed by cancerous tumors into the circulating blood stream which is supplying the tumors with oxygen and nutrients has been confirmed. The presence of such cells in the blood stream has been inferred for decades because of the spread of cancerous tumors by what has been described as the hematogenous route and on very rare occasions have been visualized in blood specimens. Recently sophisticated procedures which employ reverse transcriptase in conjunction with Polymerase Chain Reaction (PCR) have been able to detect the presence of tumor cells by their molecular signature in a significant number of patients with cancer, both when the cancer is localized and after it has spread.
An additional means of detecting circulating cancer cell employs a technology known as Fluorescent Activated Cell Sorting (FACS), such as that manufactured by Becton Dickinson and Company of Franklin Lakes, N.J. The FACS detection of circulating cancer cells involves detection of cancer cells by detecting fluorescent labeled antibodies which are directed against and bound to one or more epitopes that are present on or in cancer cells, and are not present on or in normal blood cells, and/or by detecting combinations of epitopes that are present on or in circulating normal blood cells and that may or may not be present on or in cancerous cells, or combinations of the aforesaid methods.
The FACS technology is thus based on cell highlighting, i.e., it is photometric and utilizes antibody-epitope specificity, and it cannot be used to morphologically analyze cells in situ in the FACS instrument. Both the reverse transcriptase/PCR, (the molecular method), and the FACS, (the immuno-phenotypic method), require that the origin of the tumor being sought be known in order to select for the specific molecular species or immuno-phenotypic signals. The aforesaid techniques have contributed to confirmation of the theory that cancer cells do circulate in the blood stream, but these techniques are not practical especially in point of care applications, by virtue of their cost and/or nature, for detecting the presence or absence of circulating tumorous cancer cells in the blood stream. Thus, there is no general or generic blood analyzing procedure for the detection and confirmation of the malignant nature of circulating cancer cells, regardless of their source, in a patient. In addition, neither the aforesaid molecular nor the immuno-phenotypic methods utilize in situ, i.e., in a closed sampling system, cytopathologically-based analyses to determine the morphometric characteristics of circulating cells which permit cancer cells to be identified and confirmed.
Since approximately eighty two percent of all cancers are epithelial in origin (seventy two percent of which are fatal), epithelial cancer cells should be detectable in circulating blood. While the presence of epithelial cells in the circulating blood stream does not, by itself, prove malignancy, it does alert the cytologist to the greater likelihood of malignancy since epithelial cells are not normally seen in the circulating blood stream. In certain cases, however; such as after surgery; or as a result of physical trauma; or as a result of dental flossing, or in cases of prostatitis, for example, it is possible that non-malignant epithelial cells may be found in the circulating blood stream. Visual morphological analysis of cells is currently the most reliable way to distinguish cancerous epithelial cells from benign epithelial cells which are found in the circulating blood sample. One problem which exists in connection with attempts to detect circulating cancer cells in blood via morphological analysis relates to the fact that circulating cancer cells in blood are often virtually indistinguishable from circulating hematologic progenitor cells, or blasts, by cytological analysis alone.
The paucity of cancer cells that may be present in a sample of circulating blood would require the cytopathologist to carefully examine approximately ten million nucleated blood cells in order to find one cancer cell, and that one cancer cell would be randomly located in the ten million nucleated blood cells, which in turn will themselves be homogeneously dispersed in a sea of five billion non-nucleated cellular blood constituents, i.e., the erythrocytes, plus two hundred fifty million platelets, all of which will be found in one milliliter of blood. Such a task would be very time consuming, and is thus impractical for use in analyzing a patient""s blood for the presence or absence of cancer cells.
A technique has been developed to quantitate constituent layers in a complex material mixture by centrifuging a sample of the material mixture in a capillary tube or other container which contains an insert, typically a float. The float complements the configuration of the sample container, and is preferably cylindrical when a tubular container is used. The insert has a specific gravity which causes it to settle into the centrifuged mixture to a degree which creates a spatially restricted free volume in the container into which the layer, or layers to be measured will settle. The layers to be measured are thus physically elongated, and can thus be more easily and accurately measured. The aforesaid technique is described in U.S. Pat. Nos. 4,027,660, issued Jun. 7, 1977; 4,082,085 issued Apr. 4, 1978; 4,156,570 issued May 29, 1979; and others. This technology is presently being marketed by Becton Dickinson and Company under the registered trademark xe2x80x9cQBCxe2x80x9d. This xe2x80x9cQBCxe2x80x9d technology has been adapted for use in the isolation and identification of microfilarial infestation of a blood sample, as set forth in U.S. Pat. No. 4,190,328, issued Feb. 26, 1980. U.S. Pat. Nos. 5,403,714, issued Apr. 4, 1995; 5,496,704, issued Mar. 5, 1996; 5,506,145, issued Apr. 9, 1996; and others describe the use of the aforesaid xe2x80x9cQBCxe2x80x9d technology to assay anticoagulated whole blood for various analytes; and also to assay tissue samples for the presence or absence of cancerous tumor cells, wherein tissue samples are admixed with a saline buffer solution prior to analysis.
Commonly owned co-pending U.S. patent applications Ser. No. 08/976,886, filed Nov. 24, 1997, and U.S. Ser. No. 09/507,635, filed Feb. 22, 2000 both relate to the detection of circulating cancer cells in a sample of anticoagulated whole blood. The methods described in these patent applications describe the detection of intact epithelial tumor cells in the blood sample, but do not suggest that circulating fragments of epithelial tumor cells could be detected in the blood sample.
While the aforesaid copending patent applications describe the detection of intact cancer cells in an anticoagulated whole blood sample, since intact cancer cells may not be present in a blood sample of an individual with cancer, there exists a need for a simple procedure, and a system for performing such a procedure, whereby a sample of capillary or venous blood could be quickly and accurately analyzed for the presence or absence of circulating fragments of cancer cells, or fragments of other target cells. Additionally, the procedure should enable one to differentiate cancer cell fragments from other cell fragments; and also enable one to confirm the nature of any detected cell fragments, all in situ, in the blood sampling paraphernalia.